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Bound NO in human red blood cells: fact or artifact?
Nitric Oxide 10 (2004) 221–228 www.elsevier.com/locate/yniox
Bound NO in human red blood cells: fact or artifact? Nathan S. Bryan, Tienush Rassaf,1 Juan Rodriguez, and Martin Feelisch¤ Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA 02118, USA Department of Molecular and Cellular Physiology, LSU Health Sciences Center, Shreveport, LA 71130, USA Received 27 February 2004; received in revised form 26 April 2004 Available online 28 May 2004
Abstract There has been considerable debate over the nature and chemistry of the interaction between nitric oxide (NO) and red blood cells (RBCs), in particular whether hemoglobin consumes or conserves NO bioactivity. Given the vast range of nitrosation levels reported for human RBCs in the literature, we sought to investigate whether there was a common denominator that could account for such discrepancies across diVerent methodologies and reaction conditions and if such a pathway may exist in physiology. Here, we show that there are marked diVerences in reactivity toward NO between human and rat hemoglobin, which oVers a mechanistic explanation for why basal levels of NO-adducts in primate RBCs are considerably lower than those in rodents. We further demonstrate that the inadvertent introduction of trace amounts of nitrite and incomplete thiol alkylation lead to rapid heme and thiol nitros(yl)ation, with generation of nitrosylhemoglobin (NOHb) and S-nitrosohemoglobin (SNOHb), while neither species is detectable in human RBCs at physiological nitrite concentrations. Thus, caution should be exercised in interpreting experimental results on SNOHb/NOHb levels that were obtained in the absence of knowledge about the degree of nitrite contamination, in particular when a physiological role for such species is implicated. 2004 Elsevier Inc. All rights reserved. Keywords: Nitric oxide; Nitrite; Nitrosation; Hemoglobin; Erythrocytes
Endothelium-derived nitric oxide (NO) plays an important role in vascular homeostasis by regulating blood vessel tone, and inhibiting smooth muscle cell proliferation, blood cell adhesion as well as lipid peroxidation [1]. In addition to its interactions with cells of the vascular wall, a signiWcant part of the NO produced by the endothelium comes into direct contact with blood. Given its fast rate of reaction with hemoglobin (Hb), the fate of NO was long thought to be dictated largely by its interaction with RBCs [2]. The classical paradigm of Hb as the principal NO sink (scavenger) in the cardiovascular system [3] was Wrst challenged by the notion that NO may be transported by RBCs as an S-nitroso adduct of Cys-93 in the -chain of hemoglobin, a product coined S-nitrosohemoglobin (SNOHb) [4–6]. More recently, ¤
Corresponding author. Fax: 1-617-414-8151. E-mail address: [email protected] (M. Feelisch). 1 Present address: Division of Cardiology, Pulmonary Diseases and Angiology, Department of Medicine, Heinrich-Heine-University, Duesseldorf, Germany. 1089-8603/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.niox.2004.04.008
evidence has been presented for a nitrite reductase activity of Hb that is capable of generating NO and thought to be responsible for the formation of SNOHb and NOHb in RBCs [7,8]. However, reports from these and other groups on basal levels of intracellular SNOHb have been highly inconsistent, ranging anywhere from 5 M to01 nM. Adding to the confusion, a recent study from our group using a chemiluminescence technique that was developed for trace level quantiWcation of NOadducts in complex biological matrices, failed to detect endogenous SNOHb in both, arterial and venous blood of healthy human volunteers, while in rodent blood levels similar to those reported by others were found [9]. These apparent inconsistencies led us to explore more closely what factors may account for the diVerences in SNOHb levels between diVerent species, and for the discrepancies in the reported values for humans. Given a recent report on the occurrence of similar plasma nitrite concentrations across several mammalian species, we felt it was unlikely that the diVerences in SNOHb levels in diVerent species are consequences of diVerences in body NO
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production [10], but instead might be driven by speciesdiVerences in thiol reactivity. Alternatively, and in light of recent work from our group linking nitros(yl)ation products in blood and tissues to the cellular redox status [11], we investigated whether there is a species-diVerence in glutathione (GSH) or ascorbate (Asc) levels in RBCs or plasma that dictates the resulting Hb/NO biochemistry. Another factor we considered was the possibility that trace amounts of nitrite, which is a common contaminant of aqueous buVer solutions, glass- and plasticware as well as ultraWltration membranes [12], present a problem in analytical settings aimed at investigating the fate of NO. Given the possible origins of the discrepancies in reported SNOHb values and the profound physiological consequences drawn from them, there must be a clear understanding of the underlying chemistry between Hb, nitrite, and NO, which has not been investigated in this context. In the present study, we wish to draw attention to important new Wndings that account for the varying SNOHb values based on the reactivity of Hb of diVerent species and on the artifactual formation/detection of SNOHb/NOHb during sample processing that may be interpreted as naturally occurring.
Experimental procedures Characteristics of the study population Nine men and nine women with an average age of 33 § 2 years (range 19–58 years), body weight of 70 § 3 kg, and height of 171 § 2 cm gave their informed consent before participating in the study. All volunteers were in excellent general health, non-smokers, and none were on regular medication or revealed present or past evidence of cardiovascular diseases known to aVect endothelial function such as hypertension, hypercholesterolemia, heart failure, or diabetes mellitus. In addition, Wve patients (3 men, 2 women; range 39–55 years) undergoing routine diagnostic cardiac catheterization were enrolled in the study. Male Wistar rats and guinea pigs were obtained from Harlan, housed three/cage in a ventilated micro-isolator caging system, and kept on a reversed 12/ 12 light/dark cycle. Animals were fed a standard diet and allowed access to food and water ad libidum. Experiments in animals and human subjects were approved by the Animal Care and Use Committee and the Institutional Review Board, respectively, of the Louisiana State University Health Sciences Center in Shreveport. Blood sampling and detection Blood was collected into tubes containing NEM (Merck, Germany) and EDTA (10 and 2.5 mM, respectively) to block SH-groups and inhibit transition metal-catalyzed transnitrosation reactions, preventing artiWcial nitrosation,
as well as thiolate- and ascorbate-mediated degradation of endogenous RSNOs [13]. Plasma and RBCs were obtained by centrifugation at 800g and 4°C for 10 min, and RBCs were subjected to hypotonic lysis in water containing NEM and EDTA (1:4 v/v) without further delay. In a subset of experiments, the anion exchange protein-1 and Xavoprotein inhibitors, 4,4⬘-diisothiocyanostilbene-2,2⬘-disulfonic acid (DIDS) and diphenyleneiodonium (DPI) (100M and 20 mM, respectively), as well as ferricyanide (4mM) were added to whole blood as well as to the lysis solution. Nitroso species in plasma and RBCs were quantiWed by reductive denitrosation of samples using a mixture of iodine/ iodide in glacial acetic acid and subsequent detection of the liberated NO by gas-phase chemiluminescence reaction with ozone, as described [14]. Nitrite data obtained by chemiluminescence were validated using high pressure liquid ion chromatography with on-line reduction of nitrate to nitrite and subsequent post-column derivatization with the Griess reagent [15]. Preparation of hemoglobin and standards Fresh human blood was collected in heparinized (10 U/ mL) tubes. Plasma and buVy coat were removed after centrifugation at 800g for 10 min. RBCs were washed three times in 10 mM phosphate buVer adjusted to 290 mOsm with NaCl and lysed at 1:4 with a 5 mM phosphate buVer. The lysed RBC solution was then centrifuged at 22,000g for 30 min. OxyHb was obtained by passing the upper half of the supernatant over a G-25 Sephadex column and diethylenetriamine pentaacetic acid (DTPA) was added afterwards to a Wnal concentration of 100 M. In pilot studies, the reactivity of this freshly obtained Hb with NO was compared to commercially available human Hb (Sigma), and quantitatively similar results were obtained. Rat Hb was purchased from Sigma and prepared as recently described [16]. SNOHb of either species was prepared from oxyHb by trans-nitrosation with a substoichiometric concentration of S-nitrosocysteine (molar ratio oxyHb:CysNO 3:1), and concentrations were determined using the Saville reaction. NOHb was prepared from human deoxy-Hb with NO (NO:heme ratio 1:1) in phosphate buVer under argon and concentrations were determined spectrophotometrically [17]. Dilutions of either stock solution were made fresh in oxygen-free buVer and stored for 63 h on ice in the dark. Aqueous NO solutions were prepared as described [18] and diluted in deoxygenated saline immediately before use. NO concentrations in these dilutions were determined by chemiluminescence under non-reducing conditions (injections into water), with nitrite contaminations being 62% of the NO concentration. Antioxidant measurements Plasma and blood ascorbate content was assayed essentially as described by Carr et al. [19]. After
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separation of plasma and RBC, 100 L of 50% metaphosphoric acid was added to 900 L plasma and RBC lysate, followed by vortexing and centrifugation at 14,000 rpm for 10 min. Ascorbic acid (AA) in the deproteinized samples was oxidized to dehydroascorbic acid (DHA) by addition of 25 L of 0.2% 2,6-dichlorophenolindophenol (DCIP) to 250 L sample. Following 1 h incubation at room temperature, 250 L each of 2% thiourea (in 5% metaphosphoric acid) and 2% 2,4-dinitrophenylhydrazine (DNPH) (in 12 M H2SO4) were added, and the samples were further incubated for 3 h at 60 °C. Reactions were stopped by addition of ice-cold 18 M H2SO4 (500 L) to each tube, and the content was transferred to 96-well plates for immediate reading at 524 nm using a tunable plate reader (Quant, Bio-TEK Instruments). Control samples contained H2O instead of DCIP, and deproteinization buVer was used for the blank to determine DHA concentration in each sample. An additional blank was run in which DNPH was not added until after the addition of H2SO4 to account for sample-speciWc background coloration [20]. Total ascorbate concentration in tissues was determined by the diVerence in readings of the DCIP-treated and background control samples by comparison to a standard curve with authentic ascorbate. AA concentrations were determined by the diVerence in the readings of the DCIP-treated and untreated samples. The concentration of GSH and GSSG in plasma and RBC lysate was determined using the Tietze recycling assay [21].
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human and rat RBCs, were conducted to elucidate the reason for the diVerences in SNOHb levels that have been observed across species. The results, shown in Table 1, include comparisons of the concentrations of reduced and oxidized forms of glutathione and ascorbate as indicators of the redox status in RBCs. The values reported here are consistent with those reported in other studies [22], and indicate that no major diVerences in antioxidant levels exist between the two species. The similarities in redox environment between these two species argue strongly that the diVerences in SNOHb levels are related to factors other than the cellular redox state. Comparison between Hb thiol reactivity and formation of SNOHb Following a recent report by Miranda [23] on diVerences in reactivity of the highly reactive cysteine residues of Hb in mice, rats, guinea pigs, and humans, we attempted to correlate these thiol reactivities with the endogenous concentrations of SNOHb we detected for those species. As shown in Fig. 1, there is indeed a signiW-
Data presentation Data are presented either as original recordings or the means § SEM from n individual experiments with buVer and reagent blanks subtracted from each experimental value. Results Association between redox indicators and SNOHb formation Quantitative assessments of the concentration of two key constituents of the cellular antioxidant network, in
Fig. 1. Correlation between basal concentrations of S-nitroso products in red blood cell lysate from various sources with the reactivity of highly reactive cysteine residues of hemoglobin from the same species (data taken from [22]). Means § SEM (humans, n D 13; mice, n D 8; rats, n D 9; guinea pigs, n D 6; C57 mice blood was a duplicate determination using pooled blood from four mice for each determination).
Table 1 Antioxidant status of rat and human blood GSH (M)
GSSG (M)
Asc (M)
DHA (M)
Plasma Rat Human
199.5 § 50.0 107.9 § 15.8
19.1 § 9.5 4.8 § 2.2
25.2 § 5.5 63.2 § 15.2
30.7 § 5.2 11.3 § 1.7
Red blood cells Rat Human
3130 § 750 3188 § 525
92.3 § 22.2 89.1 § 22.4
2.5 § 1.6 1.5 § 0.7
16.7 § 6.2 36.9 § 10.4
Reduced (GSH) and oxidized (GSSG) glutathione as well as ascorbate (AA) and dehydroascorbate (DHA) concentrations in plasma and red blood cells of male Wistar rats and humans. Means § SEM from four rats and six humans.
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cant correlation between thiol reactivity and SNOHb concentrations (r D 0.96). To further test the hypothesis that diVerences in the reactivity of human and rodent Hb to NOx existed, a direct in vitro comparison between rat and human Hb was conducted. Bolus NO solutions (Wnal concentration 5 M) were added (ratio NO:Hb D 1:1 (v/v)) to increasing concentrations of rat (n D 3) and human (n D 4) Hb (5, 10, 25, 100, 1000 M), respectively, mixed immediately, and formation of total nitroso/nitrosyl species (i.e., the sum of SNOHb, NNOHb, and NOHb) was subsequently determined (Fig. 2). Incubations with 100 M Hb led to the formation of 276 § 45 nM for rat Hb, whereas incubation of the same concentration of NO with human Hb led to a signiWcantly lower yield of 36 § 14 nM. Increasing human Hb concentration by a factor of 10 led only to a modest further increase in total nitroso/nitrosyl formation (83 § 8 nM). Formation of SNOHb could be inhibited by prior thiol alkylation (preincubation with NEM, 10 mM) (see arrows in Fig. 2). The fact that NEM did not fully inhibit the signal (in spite of complete thiol alkylation as measured using Ellman’s reagent) conWrms earlier Wndings that not all of the reaction products of
Fig. 2. Bolus NO solutions (Wnal concentration 5 M) were added (ratio NO:Hb D 1:1 (v/v)) to increasing concentrations of rat (n D 3) and human (n D 4) Hb (5, 10, 25, 100, and 1000 M), respectively, mixed immediately, and formation of nitroso (SNOHb and NNOHb) and nitrosyl species (NOHb) was subsequently determined. Formation of the total nitroso/nitrosyl signal was inhibited only partly by prior thiol alkylation with NEM (10 mM), indicating that Hb nitrosation occurs also at sites other than thiols.
Hb with NO are of the SNO type. Similar results were obtained for NO-bolus concentrations of 2.5 and 10 M, respectively, and when lyophilized human Hb from Sigma was used instead of freshly obtained human Hb (data not shown). To perform experiments under more physiological conditions, i.e. with continuous generation of low Xuxes as opposed to bolus addition of NO, human whole blood was incubated with the NO-donor MAHMA-NO (t1/2 2.5 min, liberating 2 NO per mole; 2.5 M; Alexis) for 30 min at room temperature on a swivelling table and formation of nitroso species and Noxides in RBCs and plasma was determined. Most of the NO released by the NO-donor was converted to plasma nitrate with only a small part being converted to SNOHb (25 § 13 nM; n D 5), consistent with previous results in human blood [24]. Stability of SNOHb/NOHb-adducts during sample preparation Our failure to detect endogenous NO-adducts in humans may have been due to a methodological problem associated with the inherent instability of such species. To check for this possibility, whole blood was injected unprocessed and within 5 s of removal from the vein directly into the reductive denitrosation mixture for analysis. This procedure produces a signal that approaches the total nitrite/nitroso content in plasma and RBCs. Direct measurements in three representative samples yielded 150 § 20 nM for human and 453 § 128 nM for rat blood. This amount is similar (albeit not identical, presumably due to incomplete recovery of nitroso species under these conditions) to that obtained after sample processing when the signals for plasma/RBC nitrite and nitroso species are combined and hematocrit as well as dilution factors are taken into account (see Table 2). However, the diVerence between rat and human whole blood injection very closely mimics the values we and others report for SNOHb species in rat RBCs (»300 nM). These experiments indicate that the total NO content in blood is preserved rather well during our sample processing. Whole blood spiked with increasing concentrations of nitrite yielded 92 § 15% recovery, indicating there is no recovery problem with our assay either (data not shown). Furthermore, the immediate injection of rat blood yields a much higher signal than that of
Table 2 Sum of the parts of NO products and metabolites in rat vs human blood
Rats Humans
Plasma Nitrosyl (nM)
Nitroso (nM)
Nitrite (nM)
Red blood cells Nitrosyl (nM)
Nitroso (nM)
Nitrite (nM)
Whole blood (Weighed sum) (nM)
01 01
4.3 § 0.3 7.2 § 1.1
156 § 23 205 § 21
10.8 § 1.8 01
266 § 51 01
530 § 123 263 § 150
483 § 76 240 § 98
The weighed sum was determined as product of (total plasma)(1-hematocrit) + (total RBC)(hematocrit). Means § SEM from 12 to 14 rats and 17 humans.
N.S. Bryan et al. / Nitric Oxide 10 (2004) 221–228
human blood (see Fig. 3). Since plasma nitrite concentrations are comparable [9,10], the diVerence can only be due to the endogenous NO species in rat blood cells that is absent in humans. Stamler et al. [25] described the export of NO bioactivity from RBCs by anion exchange protein-1 (AE1) to the RBC membrane, and Gladwin et al. [26] the stabilization of SNOHb by ferricyanide. To investigate the possible contribution of these processes to the assessment of endogenous NO-adducts in human RBCs, the AE1 inhibitor, DIDS (100 M) and the Xavoprotein inhibitor, DPI (20 mM), as well as ferricyanide (4 mM) were added to the lysis solution [25,26]. To conWrm that these compounds do in fact stabilize endogenous SNOHb, RBC-lysate was spiked with SNOHb/NOHb (i.e., a mixture of SNOHb and NOHb [13], up to 100 nM) in the presence of DPI, DIDS, and ferricyanide. This measure resulted in a 98 § 5% recovery of these species, whereas spiking experiments performed without the inhibitors resulted in a 02% recovery, suggesting that this inhibitor cocktail stabilized SNOHb during the sample processing. When applied to human whole blood, SNOHb concentrations of 100 § 30 nM were detected. Although these concentrations represent only a small fraction of the diVerences in SNOHb values reported by Stamler’s group and ours, we decided to further investigate the origin of the sudden emergence of this SNOHb signal. The results of this investigation are described below. Nitrite as a confounding factor in the detection of NOadducts With recent reports of nitrite serving as a stored pool of NO by its interactions with Hb [7,8], we sought to
Fig. 3. Original tracing of chemiluminescence signals from direct and immediate injection of rat and human whole blood into the denitrosation mixture. Reductive solution was changed after injection of rat whole blood to prevent incomplete recovery of subsequent samples due to high hemoglobin content.
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investigate if nitrite could be utilized to form NOadducts within RBCs. Considering this phenomenon and the fact that nitrite is a common source of contamination in buVers and sample preparation protocols, we sought to determine whether trace amounts of nitrite could lead to nitrosation chemistry in RBCs that would occur even at physiological pH. The above-described inhibitor cocktail was prepared to prevent the decomposition of any endogenous nitroso compound(s) in the RBC. When RBCs were lysed in a solution that contained only 4 mM ferricyanide added to NEM/EDTA/water, we detected 47 § 5 nM NO-adducts, which is in agreement with the basal levels of SNOHb in human blood reported by Gladwin et al. [26]. However, when we conducted the same experiments with hemolysate of human RBCs diluted 1:4 in lysis solution containing only water, NEM, and EDTA the values were below the detection limit of our method (1 nM bound NO). Analysis of the lysis solutions containing the above combination of inhibitors revealed that it contained 160 nM nitrite. By comparison, only 20 nM nitrite was found in our NEM/EDTA/water. This raises the question as to the eYcacy of the stabilizing eVect or the additional contamination of nitrite in the stabilizing solution that leads to the detection of a nitroso in RBCs. To systematically address this problem, known amounts of nitrite were added to the lysis solution and RBC-bound NO was subsequently detected. RBCs lysed in the presence of increasing concentrations of nitrite in the lysis solutions indeed caused nitrosation as soon as the concentrations exceeded 100 nM nitrite (see Fig. 4). For comparison, the same experiments were carried out using albumin instead of Hb, and nitrite did not lead to any detectable nitrosation. Therefore, this nitrite-mediated nitrosation appears to be due to the unique properties of Hb.
Fig. 4. SNOHb formation in human red blood cell lysates as a function of increasing concentrations of nitrite in the lysis solution. Means § SEM from 4 to 6 individual determinations with blood taken from the same donor.
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Fig. 5. Original tracing depicting incomplete nitrite scavenging by sulfanilamide due to an inadequate pre-incubation time. Complete quenching of nitrite by acidiWed sulfanilamide requires 15 min preincubation in biological samples. Any pre-incubation less than 15 min yields an NO signal that may be misinterpreted as SNOHb.
Another confounding factor noted in this study relates to the use of acidiWed sulfanilamide to quench nitrite in biological samples. This process requires adequate periods of time for pre-incubation, especially in biological samples. Extensive validation experiments in our laboratory determined that complete scavenging of nitrite by acidiWed sulfanilamide requires a minimum of 15 min, with shorter pre-incubations leading to a false positive signal due to remaining nitrite (Fig. 5). Another problem involves the temperature of the reaction mixtures used in methodologies employing reductive denitrosation by iodine/iodide and gas phase chemiluminescence for the detection of bound NO. We caution that when the temperature of the reaction vessel exceeds 60 °C, not only is nitrite incompletely recovered from heme-containing biological samples, it quickly becomes converted within the reaction mixture to a mercury stable nitroso species with the characteristics of NOHb.
Discussion The results of this study establish two important factors that account for the disparate reports published to date on the SNOHb content in blood samples. The Wrst factor is the diVerence in hemoglobin between species, as demonstrated by the strong correlation between thiol reactivity and SNOHb concentrations (r D 0.96) in Fig. 1. This correlation provides compelling evidence for marked diVerences in reactivity toward NOx between diVerent Hb species, and oVers an explanation for the diVerences reported in the literature across species. The molecular
origin of this correlation would argue for a central role for Cys-125 in the Hb protein and for a much more limited relevance for Cys-93, the active site previously associated with transport of NO in the protein. The dramatic variations of SNOHb values across species should also elicit caution in the selection of animal models for the study of nitrosation chemistry in blood. The second factor contributing to the variability of reported SNOHb values arises from artifacts generated by residual amounts of nitrite. Our results indicate that as little as 200 nM nitrite contamination in solutions used to lyse RBCs can lead to formation of artifactual NO-Hb species and complicate interpretation of results that may otherwise present as physiological conditions. Seemingly small traces of nitrite, which are present in most buVers and reagents, can cause these artiWcial nitrosation products that could be interpreted as endogenous RBC NO-adducts. For instance, experiments in which solutions were subjected to Wltration (as in many sample preparation protocols) revealed that certain Wlter materials introduce signiWcant amounts of nitrite into the buVer (50–500 nM, depending on Wlter material and Wltrated volume). Thus, for cases such as the SNOHb content in human RBCs, the apparent concentrations detected could be due exclusively to nitrite contamination. With an understanding of these two factors described above, we can begin to rationalize the diVerences in NO/ Hb reaction products reported in the literature. As reported recently, using our validated method we failed to detect any endogenous bound NO in human RBCs in arterial or venous blood whereby we consistently measure levels in rodents in agreement with other groups [9]. Even though the values we report for humans are far below those reported by other groups, the fact that the concentrations we report for rats, mice, and guinea pigs are very similar to these same groups excludes a methodological problem. We can account for and reproduce the numbers reported by others simply by the addition of nitrite directly into whole blood or by using solutions inadvertently contaminated with high nanomolar concentrations of nitrite. The fact that we can recover almost 100% of spiked SNOHb in the presence of diVerent inhibitors but only detect very low nanomolar in unspiked blood samples further strengthens our hypothesis that high concentrations of SNOHb are absent in the human circulation under normal, noninXammatory conditions. Careful accounting of all detectable species in whole blood immediately analyzed compared with individual detectable plasma and RBCs species rules out an inadequate detection or instability during sample processing using our method. Based on these Wndings, this raises the question whether basal NOadducts actually exist in human RBCs or whether they are the result of artifactual nitrosation via nitrite introduced during sample processing. Whenever nitrite
N.S. Bryan et al. / Nitric Oxide 10 (2004) 221–228
concentrations are not strictly accounted for this can in fact lead to a high degree of variability in SNOHb/ NOHb concentrations in RBCs. In agreement with data from Cosby et al. [7] and Nagababu et al. [8], we demonstrate nitrite’s ability to form NO-Hb-adducts. As we have here shown, addition of just 200 nM nitrite above the normal plasma and/or RBC concentration of nitrite is enough to reach a threshold capable of nitrosating Hb, whereas normal physiological levels fail to do so. While this nitrosation pathway is unlikely to prevail in physiology, the ability of elevated levels of nitrite to nitrosate RBCs may have important in vivo implications during inXammation or endotoxemia, in which there is excess nitrite production secondary to increased NO production. Whether these Wndings may be extrapolated to other cells or other heme-containing cell constituents remains to be investigated. Our results clearly provide a mechanistic explanation on the diVerences in basal SNOHb across several mammalian species and furthermore account for the vast ranges of values reported throughout the literature by diVerent research groups. Moreover, these data suggest that SNOHb does not form to a signiWcant degree in the human circulation but rather represents an inherent artifact that is produced during sample processing, adding a critical element to an active major debate in biochemistry and physiology. We hope this report will create awareness to the careful attention to detail during sample preparation in controlling for interfering substances and chemicals that may otherwise go unnoticed and unintentionally generate artifactual data that may be interpreted as normal physiology. Extreme care should also be taken when extrapolating results from experimental animals to humans when there are clear diVerences in target reactivity. The ease with which nitritemediated nitros(yl)ation reactions proceed at physiological pH in RBCs suggests an alternative route of NO adduct formation that may extend to other biological compartments.
Acknowledgments We thank Dr. Fumito Saijo for skillful technical assistance. This work was supported in part by National Institutes of Health Grant RO1 HL 69029 (to M.F.). T.R. is a research fellow sponsored by the Deutsche Forschungsgemeinschaft.
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conditions, Proc. Natl. Acad. Sci. USA 99 (16) (2002) 10341– 10346. [25] J.R. Pawloski, D.T. Hess, J.S. Stamler, Export of red blood cells of nitric oxide bioactivity, Nature 409 (2001) 622–626.
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Bound NO in human red blood cells: fact or artifact? Nathan S. Bryan, Tienush Rassaf,1 Juan Rodriguez, and Martin Feelisch¤ Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA 02118, USA Department of Molecular and Cellular Physiology, LSU Health Sciences Center, Shreveport, LA 71130, USA Received 27 February 2004; received in revised form 26 April 2004 Available online 28 May 2004
Abstract There has been considerable debate over the nature and chemistry of the interaction between nitric oxide (NO) and red blood cells (RBCs), in particular whether hemoglobin consumes or conserves NO bioactivity. Given the vast range of nitrosation levels reported for human RBCs in the literature, we sought to investigate whether there was a common denominator that could account for such discrepancies across diVerent methodologies and reaction conditions and if such a pathway may exist in physiology. Here, we show that there are marked diVerences in reactivity toward NO between human and rat hemoglobin, which oVers a mechanistic explanation for why basal levels of NO-adducts in primate RBCs are considerably lower than those in rodents. We further demonstrate that the inadvertent introduction of trace amounts of nitrite and incomplete thiol alkylation lead to rapid heme and thiol nitros(yl)ation, with generation of nitrosylhemoglobin (NOHb) and S-nitrosohemoglobin (SNOHb), while neither species is detectable in human RBCs at physiological nitrite concentrations. Thus, caution should be exercised in interpreting experimental results on SNOHb/NOHb levels that were obtained in the absence of knowledge about the degree of nitrite contamination, in particular when a physiological role for such species is implicated. 2004 Elsevier Inc. All rights reserved. Keywords: Nitric oxide; Nitrite; Nitrosation; Hemoglobin; Erythrocytes
Endothelium-derived nitric oxide (NO) plays an important role in vascular homeostasis by regulating blood vessel tone, and inhibiting smooth muscle cell proliferation, blood cell adhesion as well as lipid peroxidation [1]. In addition to its interactions with cells of the vascular wall, a signiWcant part of the NO produced by the endothelium comes into direct contact with blood. Given its fast rate of reaction with hemoglobin (Hb), the fate of NO was long thought to be dictated largely by its interaction with RBCs [2]. The classical paradigm of Hb as the principal NO sink (scavenger) in the cardiovascular system [3] was Wrst challenged by the notion that NO may be transported by RBCs as an S-nitroso adduct of Cys-93 in the -chain of hemoglobin, a product coined S-nitrosohemoglobin (SNOHb) [4–6]. More recently, ¤
Corresponding author. Fax: 1-617-414-8151. E-mail address: [email protected] (M. Feelisch). 1 Present address: Division of Cardiology, Pulmonary Diseases and Angiology, Department of Medicine, Heinrich-Heine-University, Duesseldorf, Germany. 1089-8603/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.niox.2004.04.008
evidence has been presented for a nitrite reductase activity of Hb that is capable of generating NO and thought to be responsible for the formation of SNOHb and NOHb in RBCs [7,8]. However, reports from these and other groups on basal levels of intracellular SNOHb have been highly inconsistent, ranging anywhere from 5 M to01 nM. Adding to the confusion, a recent study from our group using a chemiluminescence technique that was developed for trace level quantiWcation of NOadducts in complex biological matrices, failed to detect endogenous SNOHb in both, arterial and venous blood of healthy human volunteers, while in rodent blood levels similar to those reported by others were found [9]. These apparent inconsistencies led us to explore more closely what factors may account for the diVerences in SNOHb levels between diVerent species, and for the discrepancies in the reported values for humans. Given a recent report on the occurrence of similar plasma nitrite concentrations across several mammalian species, we felt it was unlikely that the diVerences in SNOHb levels in diVerent species are consequences of diVerences in body NO
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production [10], but instead might be driven by speciesdiVerences in thiol reactivity. Alternatively, and in light of recent work from our group linking nitros(yl)ation products in blood and tissues to the cellular redox status [11], we investigated whether there is a species-diVerence in glutathione (GSH) or ascorbate (Asc) levels in RBCs or plasma that dictates the resulting Hb/NO biochemistry. Another factor we considered was the possibility that trace amounts of nitrite, which is a common contaminant of aqueous buVer solutions, glass- and plasticware as well as ultraWltration membranes [12], present a problem in analytical settings aimed at investigating the fate of NO. Given the possible origins of the discrepancies in reported SNOHb values and the profound physiological consequences drawn from them, there must be a clear understanding of the underlying chemistry between Hb, nitrite, and NO, which has not been investigated in this context. In the present study, we wish to draw attention to important new Wndings that account for the varying SNOHb values based on the reactivity of Hb of diVerent species and on the artifactual formation/detection of SNOHb/NOHb during sample processing that may be interpreted as naturally occurring.
Experimental procedures Characteristics of the study population Nine men and nine women with an average age of 33 § 2 years (range 19–58 years), body weight of 70 § 3 kg, and height of 171 § 2 cm gave their informed consent before participating in the study. All volunteers were in excellent general health, non-smokers, and none were on regular medication or revealed present or past evidence of cardiovascular diseases known to aVect endothelial function such as hypertension, hypercholesterolemia, heart failure, or diabetes mellitus. In addition, Wve patients (3 men, 2 women; range 39–55 years) undergoing routine diagnostic cardiac catheterization were enrolled in the study. Male Wistar rats and guinea pigs were obtained from Harlan, housed three/cage in a ventilated micro-isolator caging system, and kept on a reversed 12/ 12 light/dark cycle. Animals were fed a standard diet and allowed access to food and water ad libidum. Experiments in animals and human subjects were approved by the Animal Care and Use Committee and the Institutional Review Board, respectively, of the Louisiana State University Health Sciences Center in Shreveport. Blood sampling and detection Blood was collected into tubes containing NEM (Merck, Germany) and EDTA (10 and 2.5 mM, respectively) to block SH-groups and inhibit transition metal-catalyzed transnitrosation reactions, preventing artiWcial nitrosation,
as well as thiolate- and ascorbate-mediated degradation of endogenous RSNOs [13]. Plasma and RBCs were obtained by centrifugation at 800g and 4°C for 10 min, and RBCs were subjected to hypotonic lysis in water containing NEM and EDTA (1:4 v/v) without further delay. In a subset of experiments, the anion exchange protein-1 and Xavoprotein inhibitors, 4,4⬘-diisothiocyanostilbene-2,2⬘-disulfonic acid (DIDS) and diphenyleneiodonium (DPI) (100M and 20 mM, respectively), as well as ferricyanide (4mM) were added to whole blood as well as to the lysis solution. Nitroso species in plasma and RBCs were quantiWed by reductive denitrosation of samples using a mixture of iodine/ iodide in glacial acetic acid and subsequent detection of the liberated NO by gas-phase chemiluminescence reaction with ozone, as described [14]. Nitrite data obtained by chemiluminescence were validated using high pressure liquid ion chromatography with on-line reduction of nitrate to nitrite and subsequent post-column derivatization with the Griess reagent [15]. Preparation of hemoglobin and standards Fresh human blood was collected in heparinized (10 U/ mL) tubes. Plasma and buVy coat were removed after centrifugation at 800g for 10 min. RBCs were washed three times in 10 mM phosphate buVer adjusted to 290 mOsm with NaCl and lysed at 1:4 with a 5 mM phosphate buVer. The lysed RBC solution was then centrifuged at 22,000g for 30 min. OxyHb was obtained by passing the upper half of the supernatant over a G-25 Sephadex column and diethylenetriamine pentaacetic acid (DTPA) was added afterwards to a Wnal concentration of 100 M. In pilot studies, the reactivity of this freshly obtained Hb with NO was compared to commercially available human Hb (Sigma), and quantitatively similar results were obtained. Rat Hb was purchased from Sigma and prepared as recently described [16]. SNOHb of either species was prepared from oxyHb by trans-nitrosation with a substoichiometric concentration of S-nitrosocysteine (molar ratio oxyHb:CysNO 3:1), and concentrations were determined using the Saville reaction. NOHb was prepared from human deoxy-Hb with NO (NO:heme ratio 1:1) in phosphate buVer under argon and concentrations were determined spectrophotometrically [17]. Dilutions of either stock solution were made fresh in oxygen-free buVer and stored for 63 h on ice in the dark. Aqueous NO solutions were prepared as described [18] and diluted in deoxygenated saline immediately before use. NO concentrations in these dilutions were determined by chemiluminescence under non-reducing conditions (injections into water), with nitrite contaminations being 62% of the NO concentration. Antioxidant measurements Plasma and blood ascorbate content was assayed essentially as described by Carr et al. [19]. After
N.S. Bryan et al. / Nitric Oxide 10 (2004) 221–228
separation of plasma and RBC, 100 L of 50% metaphosphoric acid was added to 900 L plasma and RBC lysate, followed by vortexing and centrifugation at 14,000 rpm for 10 min. Ascorbic acid (AA) in the deproteinized samples was oxidized to dehydroascorbic acid (DHA) by addition of 25 L of 0.2% 2,6-dichlorophenolindophenol (DCIP) to 250 L sample. Following 1 h incubation at room temperature, 250 L each of 2% thiourea (in 5% metaphosphoric acid) and 2% 2,4-dinitrophenylhydrazine (DNPH) (in 12 M H2SO4) were added, and the samples were further incubated for 3 h at 60 °C. Reactions were stopped by addition of ice-cold 18 M H2SO4 (500 L) to each tube, and the content was transferred to 96-well plates for immediate reading at 524 nm using a tunable plate reader (Quant, Bio-TEK Instruments). Control samples contained H2O instead of DCIP, and deproteinization buVer was used for the blank to determine DHA concentration in each sample. An additional blank was run in which DNPH was not added until after the addition of H2SO4 to account for sample-speciWc background coloration [20]. Total ascorbate concentration in tissues was determined by the diVerence in readings of the DCIP-treated and background control samples by comparison to a standard curve with authentic ascorbate. AA concentrations were determined by the diVerence in the readings of the DCIP-treated and untreated samples. The concentration of GSH and GSSG in plasma and RBC lysate was determined using the Tietze recycling assay [21].
223
human and rat RBCs, were conducted to elucidate the reason for the diVerences in SNOHb levels that have been observed across species. The results, shown in Table 1, include comparisons of the concentrations of reduced and oxidized forms of glutathione and ascorbate as indicators of the redox status in RBCs. The values reported here are consistent with those reported in other studies [22], and indicate that no major diVerences in antioxidant levels exist between the two species. The similarities in redox environment between these two species argue strongly that the diVerences in SNOHb levels are related to factors other than the cellular redox state. Comparison between Hb thiol reactivity and formation of SNOHb Following a recent report by Miranda [23] on diVerences in reactivity of the highly reactive cysteine residues of Hb in mice, rats, guinea pigs, and humans, we attempted to correlate these thiol reactivities with the endogenous concentrations of SNOHb we detected for those species. As shown in Fig. 1, there is indeed a signiW-
Data presentation Data are presented either as original recordings or the means § SEM from n individual experiments with buVer and reagent blanks subtracted from each experimental value. Results Association between redox indicators and SNOHb formation Quantitative assessments of the concentration of two key constituents of the cellular antioxidant network, in
Fig. 1. Correlation between basal concentrations of S-nitroso products in red blood cell lysate from various sources with the reactivity of highly reactive cysteine residues of hemoglobin from the same species (data taken from [22]). Means § SEM (humans, n D 13; mice, n D 8; rats, n D 9; guinea pigs, n D 6; C57 mice blood was a duplicate determination using pooled blood from four mice for each determination).
Table 1 Antioxidant status of rat and human blood GSH (M)
GSSG (M)
Asc (M)
DHA (M)
Plasma Rat Human
199.5 § 50.0 107.9 § 15.8
19.1 § 9.5 4.8 § 2.2
25.2 § 5.5 63.2 § 15.2
30.7 § 5.2 11.3 § 1.7
Red blood cells Rat Human
3130 § 750 3188 § 525
92.3 § 22.2 89.1 § 22.4
2.5 § 1.6 1.5 § 0.7
16.7 § 6.2 36.9 § 10.4
Reduced (GSH) and oxidized (GSSG) glutathione as well as ascorbate (AA) and dehydroascorbate (DHA) concentrations in plasma and red blood cells of male Wistar rats and humans. Means § SEM from four rats and six humans.
224
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cant correlation between thiol reactivity and SNOHb concentrations (r D 0.96). To further test the hypothesis that diVerences in the reactivity of human and rodent Hb to NOx existed, a direct in vitro comparison between rat and human Hb was conducted. Bolus NO solutions (Wnal concentration 5 M) were added (ratio NO:Hb D 1:1 (v/v)) to increasing concentrations of rat (n D 3) and human (n D 4) Hb (5, 10, 25, 100, 1000 M), respectively, mixed immediately, and formation of total nitroso/nitrosyl species (i.e., the sum of SNOHb, NNOHb, and NOHb) was subsequently determined (Fig. 2). Incubations with 100 M Hb led to the formation of 276 § 45 nM for rat Hb, whereas incubation of the same concentration of NO with human Hb led to a signiWcantly lower yield of 36 § 14 nM. Increasing human Hb concentration by a factor of 10 led only to a modest further increase in total nitroso/nitrosyl formation (83 § 8 nM). Formation of SNOHb could be inhibited by prior thiol alkylation (preincubation with NEM, 10 mM) (see arrows in Fig. 2). The fact that NEM did not fully inhibit the signal (in spite of complete thiol alkylation as measured using Ellman’s reagent) conWrms earlier Wndings that not all of the reaction products of
Fig. 2. Bolus NO solutions (Wnal concentration 5 M) were added (ratio NO:Hb D 1:1 (v/v)) to increasing concentrations of rat (n D 3) and human (n D 4) Hb (5, 10, 25, 100, and 1000 M), respectively, mixed immediately, and formation of nitroso (SNOHb and NNOHb) and nitrosyl species (NOHb) was subsequently determined. Formation of the total nitroso/nitrosyl signal was inhibited only partly by prior thiol alkylation with NEM (10 mM), indicating that Hb nitrosation occurs also at sites other than thiols.
Hb with NO are of the SNO type. Similar results were obtained for NO-bolus concentrations of 2.5 and 10 M, respectively, and when lyophilized human Hb from Sigma was used instead of freshly obtained human Hb (data not shown). To perform experiments under more physiological conditions, i.e. with continuous generation of low Xuxes as opposed to bolus addition of NO, human whole blood was incubated with the NO-donor MAHMA-NO (t1/2 2.5 min, liberating 2 NO per mole; 2.5 M; Alexis) for 30 min at room temperature on a swivelling table and formation of nitroso species and Noxides in RBCs and plasma was determined. Most of the NO released by the NO-donor was converted to plasma nitrate with only a small part being converted to SNOHb (25 § 13 nM; n D 5), consistent with previous results in human blood [24]. Stability of SNOHb/NOHb-adducts during sample preparation Our failure to detect endogenous NO-adducts in humans may have been due to a methodological problem associated with the inherent instability of such species. To check for this possibility, whole blood was injected unprocessed and within 5 s of removal from the vein directly into the reductive denitrosation mixture for analysis. This procedure produces a signal that approaches the total nitrite/nitroso content in plasma and RBCs. Direct measurements in three representative samples yielded 150 § 20 nM for human and 453 § 128 nM for rat blood. This amount is similar (albeit not identical, presumably due to incomplete recovery of nitroso species under these conditions) to that obtained after sample processing when the signals for plasma/RBC nitrite and nitroso species are combined and hematocrit as well as dilution factors are taken into account (see Table 2). However, the diVerence between rat and human whole blood injection very closely mimics the values we and others report for SNOHb species in rat RBCs (»300 nM). These experiments indicate that the total NO content in blood is preserved rather well during our sample processing. Whole blood spiked with increasing concentrations of nitrite yielded 92 § 15% recovery, indicating there is no recovery problem with our assay either (data not shown). Furthermore, the immediate injection of rat blood yields a much higher signal than that of
Table 2 Sum of the parts of NO products and metabolites in rat vs human blood
Rats Humans
Plasma Nitrosyl (nM)
Nitroso (nM)
Nitrite (nM)
Red blood cells Nitrosyl (nM)
Nitroso (nM)
Nitrite (nM)
Whole blood (Weighed sum) (nM)
01 01
4.3 § 0.3 7.2 § 1.1
156 § 23 205 § 21
10.8 § 1.8 01
266 § 51 01
530 § 123 263 § 150
483 § 76 240 § 98
The weighed sum was determined as product of (total plasma)(1-hematocrit) + (total RBC)(hematocrit). Means § SEM from 12 to 14 rats and 17 humans.
N.S. Bryan et al. / Nitric Oxide 10 (2004) 221–228
human blood (see Fig. 3). Since plasma nitrite concentrations are comparable [9,10], the diVerence can only be due to the endogenous NO species in rat blood cells that is absent in humans. Stamler et al. [25] described the export of NO bioactivity from RBCs by anion exchange protein-1 (AE1) to the RBC membrane, and Gladwin et al. [26] the stabilization of SNOHb by ferricyanide. To investigate the possible contribution of these processes to the assessment of endogenous NO-adducts in human RBCs, the AE1 inhibitor, DIDS (100 M) and the Xavoprotein inhibitor, DPI (20 mM), as well as ferricyanide (4 mM) were added to the lysis solution [25,26]. To conWrm that these compounds do in fact stabilize endogenous SNOHb, RBC-lysate was spiked with SNOHb/NOHb (i.e., a mixture of SNOHb and NOHb [13], up to 100 nM) in the presence of DPI, DIDS, and ferricyanide. This measure resulted in a 98 § 5% recovery of these species, whereas spiking experiments performed without the inhibitors resulted in a 02% recovery, suggesting that this inhibitor cocktail stabilized SNOHb during the sample processing. When applied to human whole blood, SNOHb concentrations of 100 § 30 nM were detected. Although these concentrations represent only a small fraction of the diVerences in SNOHb values reported by Stamler’s group and ours, we decided to further investigate the origin of the sudden emergence of this SNOHb signal. The results of this investigation are described below. Nitrite as a confounding factor in the detection of NOadducts With recent reports of nitrite serving as a stored pool of NO by its interactions with Hb [7,8], we sought to
Fig. 3. Original tracing of chemiluminescence signals from direct and immediate injection of rat and human whole blood into the denitrosation mixture. Reductive solution was changed after injection of rat whole blood to prevent incomplete recovery of subsequent samples due to high hemoglobin content.
225
investigate if nitrite could be utilized to form NOadducts within RBCs. Considering this phenomenon and the fact that nitrite is a common source of contamination in buVers and sample preparation protocols, we sought to determine whether trace amounts of nitrite could lead to nitrosation chemistry in RBCs that would occur even at physiological pH. The above-described inhibitor cocktail was prepared to prevent the decomposition of any endogenous nitroso compound(s) in the RBC. When RBCs were lysed in a solution that contained only 4 mM ferricyanide added to NEM/EDTA/water, we detected 47 § 5 nM NO-adducts, which is in agreement with the basal levels of SNOHb in human blood reported by Gladwin et al. [26]. However, when we conducted the same experiments with hemolysate of human RBCs diluted 1:4 in lysis solution containing only water, NEM, and EDTA the values were below the detection limit of our method (1 nM bound NO). Analysis of the lysis solutions containing the above combination of inhibitors revealed that it contained 160 nM nitrite. By comparison, only 20 nM nitrite was found in our NEM/EDTA/water. This raises the question as to the eYcacy of the stabilizing eVect or the additional contamination of nitrite in the stabilizing solution that leads to the detection of a nitroso in RBCs. To systematically address this problem, known amounts of nitrite were added to the lysis solution and RBC-bound NO was subsequently detected. RBCs lysed in the presence of increasing concentrations of nitrite in the lysis solutions indeed caused nitrosation as soon as the concentrations exceeded 100 nM nitrite (see Fig. 4). For comparison, the same experiments were carried out using albumin instead of Hb, and nitrite did not lead to any detectable nitrosation. Therefore, this nitrite-mediated nitrosation appears to be due to the unique properties of Hb.
Fig. 4. SNOHb formation in human red blood cell lysates as a function of increasing concentrations of nitrite in the lysis solution. Means § SEM from 4 to 6 individual determinations with blood taken from the same donor.
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Fig. 5. Original tracing depicting incomplete nitrite scavenging by sulfanilamide due to an inadequate pre-incubation time. Complete quenching of nitrite by acidiWed sulfanilamide requires 15 min preincubation in biological samples. Any pre-incubation less than 15 min yields an NO signal that may be misinterpreted as SNOHb.
Another confounding factor noted in this study relates to the use of acidiWed sulfanilamide to quench nitrite in biological samples. This process requires adequate periods of time for pre-incubation, especially in biological samples. Extensive validation experiments in our laboratory determined that complete scavenging of nitrite by acidiWed sulfanilamide requires a minimum of 15 min, with shorter pre-incubations leading to a false positive signal due to remaining nitrite (Fig. 5). Another problem involves the temperature of the reaction mixtures used in methodologies employing reductive denitrosation by iodine/iodide and gas phase chemiluminescence for the detection of bound NO. We caution that when the temperature of the reaction vessel exceeds 60 °C, not only is nitrite incompletely recovered from heme-containing biological samples, it quickly becomes converted within the reaction mixture to a mercury stable nitroso species with the characteristics of NOHb.
Discussion The results of this study establish two important factors that account for the disparate reports published to date on the SNOHb content in blood samples. The Wrst factor is the diVerence in hemoglobin between species, as demonstrated by the strong correlation between thiol reactivity and SNOHb concentrations (r D 0.96) in Fig. 1. This correlation provides compelling evidence for marked diVerences in reactivity toward NOx between diVerent Hb species, and oVers an explanation for the diVerences reported in the literature across species. The molecular
origin of this correlation would argue for a central role for Cys-125 in the Hb protein and for a much more limited relevance for Cys-93, the active site previously associated with transport of NO in the protein. The dramatic variations of SNOHb values across species should also elicit caution in the selection of animal models for the study of nitrosation chemistry in blood. The second factor contributing to the variability of reported SNOHb values arises from artifacts generated by residual amounts of nitrite. Our results indicate that as little as 200 nM nitrite contamination in solutions used to lyse RBCs can lead to formation of artifactual NO-Hb species and complicate interpretation of results that may otherwise present as physiological conditions. Seemingly small traces of nitrite, which are present in most buVers and reagents, can cause these artiWcial nitrosation products that could be interpreted as endogenous RBC NO-adducts. For instance, experiments in which solutions were subjected to Wltration (as in many sample preparation protocols) revealed that certain Wlter materials introduce signiWcant amounts of nitrite into the buVer (50–500 nM, depending on Wlter material and Wltrated volume). Thus, for cases such as the SNOHb content in human RBCs, the apparent concentrations detected could be due exclusively to nitrite contamination. With an understanding of these two factors described above, we can begin to rationalize the diVerences in NO/ Hb reaction products reported in the literature. As reported recently, using our validated method we failed to detect any endogenous bound NO in human RBCs in arterial or venous blood whereby we consistently measure levels in rodents in agreement with other groups [9]. Even though the values we report for humans are far below those reported by other groups, the fact that the concentrations we report for rats, mice, and guinea pigs are very similar to these same groups excludes a methodological problem. We can account for and reproduce the numbers reported by others simply by the addition of nitrite directly into whole blood or by using solutions inadvertently contaminated with high nanomolar concentrations of nitrite. The fact that we can recover almost 100% of spiked SNOHb in the presence of diVerent inhibitors but only detect very low nanomolar in unspiked blood samples further strengthens our hypothesis that high concentrations of SNOHb are absent in the human circulation under normal, noninXammatory conditions. Careful accounting of all detectable species in whole blood immediately analyzed compared with individual detectable plasma and RBCs species rules out an inadequate detection or instability during sample processing using our method. Based on these Wndings, this raises the question whether basal NOadducts actually exist in human RBCs or whether they are the result of artifactual nitrosation via nitrite introduced during sample processing. Whenever nitrite
N.S. Bryan et al. / Nitric Oxide 10 (2004) 221–228
concentrations are not strictly accounted for this can in fact lead to a high degree of variability in SNOHb/ NOHb concentrations in RBCs. In agreement with data from Cosby et al. [7] and Nagababu et al. [8], we demonstrate nitrite’s ability to form NO-Hb-adducts. As we have here shown, addition of just 200 nM nitrite above the normal plasma and/or RBC concentration of nitrite is enough to reach a threshold capable of nitrosating Hb, whereas normal physiological levels fail to do so. While this nitrosation pathway is unlikely to prevail in physiology, the ability of elevated levels of nitrite to nitrosate RBCs may have important in vivo implications during inXammation or endotoxemia, in which there is excess nitrite production secondary to increased NO production. Whether these Wndings may be extrapolated to other cells or other heme-containing cell constituents remains to be investigated. Our results clearly provide a mechanistic explanation on the diVerences in basal SNOHb across several mammalian species and furthermore account for the vast ranges of values reported throughout the literature by diVerent research groups. Moreover, these data suggest that SNOHb does not form to a signiWcant degree in the human circulation but rather represents an inherent artifact that is produced during sample processing, adding a critical element to an active major debate in biochemistry and physiology. We hope this report will create awareness to the careful attention to detail during sample preparation in controlling for interfering substances and chemicals that may otherwise go unnoticed and unintentionally generate artifactual data that may be interpreted as normal physiology. Extreme care should also be taken when extrapolating results from experimental animals to humans when there are clear diVerences in target reactivity. The ease with which nitritemediated nitros(yl)ation reactions proceed at physiological pH in RBCs suggests an alternative route of NO adduct formation that may extend to other biological compartments.
Acknowledgments We thank Dr. Fumito Saijo for skillful technical assistance. This work was supported in part by National Institutes of Health Grant RO1 HL 69029 (to M.F.). T.R. is a research fellow sponsored by the Deutsche Forschungsgemeinschaft.
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